Figure 5.

EMSA analysis of the sequence spanning the DIS motif predicted to include binding sites for C/EBP and AP-1 factors. (a) Sequences from nucleotides + 243 to + 274 in the GLS region. The sequence from subtype A1 and related CRFs as a probe (DIS-A1/CRFs). The sequence used as a mutated competitor (DIS_B) includes a perfect match for the C/EBP canonical motif. DIS-A1/CRFs was incubated in the absence of nuclear proteins (b lane 1) or with 5 µg of HeLa 4-h serum response nuclear extract (b lanes 2–9 and c lane 1–4). (b) The observed DNA–protein complex (lane 2) was displaced by the cold probe (lane 3) and by the mutated competitor (lane 5), indicating a positive competition. In contrast, there was no disruption of the DNA–protein complex in the presence of a non-specific competitor (lane 4). The presence of AP-1 proteins within the DNA-proteins complex was indicated by the binding of recombinant c-Fos protein (lane 7) and the supershift reaction with anti c-Fos (lane 6) and c-Jun (lane 9) antibodies. (c) In a continuation gel run in parallel, we observed the lack of a supershift in the presence of anti-C/EBP α (lane 1) and β (lane 2) antibodies. This suggests the absence of binding sites for these proteins on the oligonucleotide used as a probe. Also, we noted that the amount of probe used was significantly consumed in the reaction when compounds such as recombinant protein and/or antibodies were added. However, no band shift was formed between the probe and the antibodies alone (b lanes 6–9, c lanes 1–4 and supplementary information S1 Fig. 5). This could suggest an interaction between AP-1 related antibodies and the probe. However, in this experiment no complex was formed between the probe and the antibody alone (supplementary information S1 Fig. 5). To improve the conciseness and clarity, the image was cropped and the bands of interest that were non-adjacent in the original gel (Supplementary information S1 Fig. 5) were juxtaposed with a clear separation.